Many blood-ingesting pests are known to feed on humans and animals, and many pests are vectors for pathogenic microorganisms which threaten human and animal health, including commercially important livestock, pets and other animals. Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodium, the protozoan which causes malaria, a devastating disease which results in approximately 1 million deaths annually. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other arboviruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis and filarial worms. The common house mosquito also transmits Wuchereria bancrofti and Brugia malayi, which cause various forms of lymphatic filariasis, including elephantiasis. Trypanasomacruzi, the causative agent of Chagas"" disease, is transmitted by various species of blood-ingesting Triatominae bugs. The tsetse fly (Glossina spp.) transmits African trypanosomal diseases of humans and cattle. Many other diseases are transmitted by various blood-ingesting pest species. The order Diptera contains a large number of blood-ingesting and disease-bearing pests, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies.
Various pesticides have been employed in efforts to control or eradicate populations of disease-bearing pests, such as disease-bearing blood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-pest organisms.
Another common class of pesticides is the organophosphates, which is perhaps the largest and most versatile class of pesticides. Organophosphates include, for example, parathion, Malathion(trademark), diazinon, naled, methyl parathion, and dichlorvos. Organophosphates are generally much more toxic than the chlorinated hydrocarbons. Their pesticidal effect results from their ability to inhibit the enzyme cholinesterase, an essential enzyme in the functioning of the insect nervous system. However, they also have toxic effects on many animals, including humans.
The carbamates, a relatively new group of pesticides, include such compounds as carbamyl, methomyl, and carbofuran. These compounds are rapidly detoxified and eliminated from animal tissues. Their toxicity is thought to involve a mechanism similar to the mechanism of the organophosphates; consequently, they exhibit similar shortcomings, including animal toxicity.
A major problem in pest control results from the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally-occurring mutants possessing biochemical, physiological or behavioristic factors that enable the pests to tolerate the pesticide. Species of Anopheles mosquitoes, for example, have been known to develop resistance to DDT and dieldrin. DDT substitutes, such as Malathion(trademark), propoxur and fenitrothion are available; however, the cost of these substitutes is much greater than the cost of DDT.
There is clearly a longstanding need in the art for pesticidal compounds that are pest-specific, that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible in the sense that they are biodegradable, and are not toxic to non-pest organisms, and have reduced or no tendency to bioaccummulate.
Many pests, including for example blood-inbibing pests, must consume and digest a proteinaceous meal to acquire sufficient essential amino acids for growth, development and the production of mature eggs. Adult pests, such as adult mosquitoes, need these essential amino acids for the production of vitellogenins by the fat body. These vitellogenins are precursors to yolk proteins which are critical components of oogenesis. Many pests, such as house flies and mosquitoes, produce oostatic hormones that inhibit egg development by inhibiting digestion of the protein meal, and thereby limiting the availability of the essential amino acids necessary for egg development.
Serine esterases such as trypsin and trypsin-like enzymes (collectively referred to herein as xe2x80x9cTTLExe2x80x9d) are important components of the digestion of proteins by insects. In the mosquito, Aedes aegypti, an early trypsin that is found in the midgut of newly emerged females is replaced, following the blood meal, by a late trypsin. A female mosquito typically weighs about 2 mg and produces 4 to 6 xcexcg of trypsin within several hours after a ingesting blood meal. Continuous boisynthesis at this rate would exhaust the available metabolic energy of a female mosquito; as a result, the mosquito would be unable to produce mature eggs, or even to find an oviposition site. To conserve metabolic energy, the mosquito regulates TTLE biosynthesis with a peptide hormone named Trypsin Modulating Oostatic Factor (TMOF). TMOF mosquitoes produce in the follicular epithelium of the ovary 12-35 hours after a blood meal; TMOF is then released into the hemolymph where it binds to a specific receptor on the midgut epithelial cells, signaling the termination of TTLE biosynthesis.
This regulatory mechanism is not unique for mosquitoes; flesh flies, fleas, sand flies, house flies, dog flies and other insect pests which need protein as part of their diet have similar regulatory mechanisms.
In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosed that injection of a hormone preparation into the body cavity of blood imbibed mosquitoes caused inhibition of egg development and sterility (Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349). Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins. Biochem. Physiol. 7:187-210) reported that injection or passage of a peptide hormone reparation into mosquitoes inhibited the TTLE biosynthesis in the epithelial cells of the gut. This inhibition caused inefficient digestion of the blood meal and a reduction in the availability of essential amino acids translocated by the hemolymph, resulting in arrested egg development in the treated insect. Borovsky observed that this inhibition of egg development does not occur when the oostatic hormone peptides are inside the lumen of the gut or other parts of the digestive system (Borovsky, D. [1988], supra).
Following the 1985 report, the isolated hormone, (a ten amino acid peptide) and two TMOF analogues were disclosed in U.S. Pat. No. 5,011,909 and 5,130,253, and in a 1990 publication (Borovsky et al. [1990]FASEB J. 4:3015-3020).Additionally, U.S. Pat. No. 5,358,934 discloses truncated forms of the full length TMOF which have prolines removed from the carboxy terminus, including the peptides YDPAP, YDPAPP, YDPAPPP, and YDPAPPPP.
Neuropeptides Y (NPY) are an abundant family of peptides that are widely distributed in the central nervous system of vertebrates. NPY peptides have also been recently isolated and identified in a cestode, a turbellarian, and in terrestrial and marine molluscs (Maule et al., 1991 xe2x80x9cNeuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide from Moniezia expansa (Cestoda: Cyclophylidea)xe2x80x9d Parasitology 102:309-316; Curry et al., 1992 xe2x80x9cNeuropeptide F: Primary Structure from the Turbellarian, Arthioposthia triangulataxe2x80x9d Comp. Biochem. Physiol. 101C:269-274; Leung et al., 1992 xe2x80x9cThe Primary Structure of Neuropeptide F (NPF) from the Garden Snail, Helix aspersaxe2x80x9d Regul. Pep. 41:71-81; Rajpara et al., 1992 xe2x80x9cIdentification and Molecular Cloning of Neuropeptide Y Homolog that Produces Prolonged Inhibition in Aplysia Neuronsxe2x80x9d Neuron. 9:505-513).
Invertebrate NPYs are highly homologous to vertebrate NPYs. The major difference between vertebrate and invertebrate NPYs occurs at the C-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereas invertebrates have an amidated phenylalanine (F). Because of this difference, the invertebrate peptides are referred to as NPF peptides.
Cytoimmunochemical analyses of NPY peptides suggest that they are concentrated in the brain of various insects, including the Colorado potato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 xe2x80x9cDistinct Localization of FMRF amide- and Bovine Pancreatic Polypeptide-Like Material in the Brain, Retrocerebal Complex and Subesophageal Ganglion of the Cockroach Periplaneta americanaxe2x80x9d L. Brain Res. 348:331-338; Veenstra et al., 1985 xe2x80x9cImmunocytochemical Localization of Peptidergic Neurons and Neurosecretory Cells in the Neuro-Endocrine System of the Colorado Potato Beetle with Antisera to Vertebrate Regulatory Peptidesxe2x80x9d Histochemistry 82:9-18). Partial purification of NPY peptides in insects suggests that both NPY and NPF are synthesized in insects (Duve et al., 1981 xe2x80x9cIsolation and Partial Characterization of Pancreatic Polypeptide-like Material in the Brain of the Blowfly Alliphora vomitoriaxe2x80x9d Biochem. J. 197, 767-770).
Researchers have recently isolated two neuropeptides with NPF-like immunoreactivity from brain extracts of the Colorado potato beetle. The researchers purified the peptides using C18 reversed phase high pressure liquid chromatography (HPLC), and determined their structure using mass spectrometry. The deduced structures of these peptides are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amid (SEQ ID NO. 1) and Ala-Pro-Ser-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF I and NPF II, respectively (Spittaels, Kurt, Peter Verhaert, Chris Shaw, Richard N. Johnston et al. [1996] Insect Biochem. Molec. Biol. 26(4):375-382).
The subject invention concerns novel pesticidal polypeptides and other compounds. In a preferred embodiment, the pesticidal agents of the subject invention (collectively referred to herein as xe2x80x9cpesticidal compoundsxe2x80x9d) inhibit digestion in pests by terminating or otherwise blocking synthesis of digestive enzymes by activating a TMOF receptor. The pesticidal polypeptides and other compounds of the present invention are usefully employed in the control of pests, such as mosquitoes, which ingest blood.
In one aspect, the pesticidal compounds of the present invention comprise novel polypeptides comprising an amino acid sequence having the formula:
A1A2A3A4A5Fxe2x80x83xe2x80x83(Formula I)
wherein:
A1 is selected from the group consisting of Y, A, D, F, G, M, P, S, and Y;
A2 is selected from the group consisting of A, D, E, F, G, N, P, S, and Y;
A3 is optionally present and is selected from the group consisting of A, D, F, G, L, P, S, and Y;
A4 is optionally present when A3 is present and is selected from the group consisting of A, F, G, L, and Y;
A5 is optionally present when A4 is present and is selected from the group consisting of A, F, L, and P;
F is a flanking region which is optionally present and is selected from the group consisting of P, PP, PPP, PPPP, and PPPPP.
The pesticidal compound preferably does not consist of YDPAP6, DYPAP6, PAP6, YDPAP, YDPAP2, YDPAP3, YDPAP4, NPTNLH, or DF-OMe.
In a narrower aspect, the pesticidal compound comprises a polypeptide having an amino acid sequence which consists essentially of the amino acid sequence of Formula I. In a preferred aspect, where the amino acid sequence is a TMOF fragment, the pesticidal compound lacks TMOF amino acids adjacent to the TMOF fragment. Instill another aspect, the peptide consists of the amino acid sequence of Formula I.
In another aspect, only A1, A2, A3, A4, and F of Formula I are present and F is attached to A4. In yet another aspect, only A1, A2, A3, and A4 of Formula I are present. In still a further aspect, only A1, A2, A3, and F of Formula I are present and F is attached to A4. In an additional aspect, only A1, A2, and A3 of Formula I are present. In another aspect, only A1, A2, and F of Formula I are present and F is attached to A2, and in a further aspect, only A1 and A2 of Formula I are present. In a preferred mode, A and D are present in the amino acid sequence and, more preferably, A, D, and Y are present.
One embodiment of the subject invention concerns a peptide comprising an amino acid sequence having the formula A1A2 (Formula II), wherein A1 is an amino acid residue selected from the group consisting of A, D, F, M, and Y, and A2 is an amino acid residue selected from the group consisting of A, D, E, P, and Y. In a preferred embodiment, the subject invention is directed to peptides which comprise the amino acids A, D, and Y.
The pesticidal compounds of the present invention have advantageous biological activity against pests. The novel polypeptides and other compounds of the invention are particularly active against blood-sucking insects, particularly against species of mosquitoes such as Aedes aegypti that are common vectors of arthropod-borne viral diseases, such as arboviruses. Other biting pests such as flies, fleas, ticks, and lice can also be controlled using peptides and methods of the subject invention. These pests utilize as their primary blood-digesting enzymes.
The subject peptides can also be used to control pests of agricultural crops. These pests include, for example, coleopterans (beetles), lepidopterans (caterpillars), and mites. The compounds of the subject invention can also be used to control household pests including, but not limited to, ants and cockroaches.
A further aspect of the subject invention are addition salts, complexes, or prodrugs such as esters of the peptides described herein, especially the nontoxic pharmaceutically or agriculturally acceptable acid addition salts. The acid addition salts can be prepared using standard procedures in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ethanedisulfonic or methanesulfonic acids. Esterification to form derivatives such as the methyl or ethyl esters, can also be performed using standard procedures.
The N-terminus and C-terminus of the pesticidal polypeptides can be blocked to further inhibit proteolysis by metabolic enzymes. Derivation of peptides to block the N-terminus or C-terminus is known in the art. For example, the N-terminus can be acetylated by methods known to those of ordinary skill in the art, and/or the C-terminus can be amidated as is well known in the art.
Peptides containing the above sequences in which only conservative substitutions have been made are also provided by the present invention. Analogues of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analogue having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally, to provide a sulfhydryl group for disulfide bond formation), are also provided.
The pesticidal polypeptides and other compounds of the present invention may also comprise D-conformation amino acids which can inhibit the ability of proteases to degrade the polypeptides. Also, derivation of the pesticidal compounds with long chain hydrocarbons will facilitate passage through the cuticle into the pest body cavity. Therefore, in a further embodiment, the subject invention provides compositions comprising the pesticidal polypeptides bound to lipids or other carriers.
Yet another aspect of the subject invention pertains to DNA sequences encoding the pesticidal polypeptides disclosed herein. These DNA sequences can readily be synthesized by a person skilled in the art, and can be used to transform an appropriate prokaryotic or eukaryotic host to enable the host to express the novel peptides. Hosts of particular interest include bacteria, yeasts, viruses, and plants. Furthermore, viruses may also be modified to transmit polynucleotides encoding the pesticidal polypeptides of the present invention. For each of these hosts, the DNA sequences may be specifically designed by a person skilled in the art to utilize codons known to be optimally expressed in the particular hosts. Advantageous promoters can also easily be employed in the polynucleotide sequences. Bacteria, yeasts, plants, and viruses each may be used in the production of pesticidal polypeptides for further use, or these hosts can be used as vehicles for direct application of the pesticidal polypeptides to the target pest. Plants can be transformed to render them toxic to a target pest species which feeds on the transformed plant. Methods for transforming plant cells utilizing, for example, Agrobacteria, are well known to those skilled in the art.
Another aspect of the subject invention pertains to a method for controlling pests comprising administering to said pest an effective amount of a pesticidal polypeptide compound of the subject invention.
The subject invention provides pest control compositions comprising pesticidal compounds and a suitable pesticidal carrier. The pest control compositions are formulated for application to the target pests or their situs. In a specific embodiment, the present invention provides recombinant hosts transformed to express a pesticidal polypeptide. The recombinant host may be, for example, prokaryotic or eukaryotic cells such as yeast or algae which have been transformed to express a pesticidal compound of the subject invention. The transformed hosts can be applied to pest habitats, such as bodies of water inhibited by mosquito larvae. Ingestion of the transformed host by a pest species in control of the pest by the pesticidal polypeptide.
In a preferred embodiment for the control of agricultural pests, the subject invention provides transformed plants which express a pesticidal polypeptide. Pest control is achieved when the pest ingests the transformed plant material.
The methods and materials of the subject invention provide a novel approach to controlling insects and insect-transmitted diseases. The peptides of the subject invention have advantageous activity and increased resistance to proteolysis over previously disclosed compounds.
As used herein, the term xe2x80x9cpesticidally effectivexe2x80x9d is used to indicate an amount or concentration of a pesticidal compound which is sufficient to reduce the number of pests in a geographical locus as compared to a corresponding geographical locus in the absence of the amount or concentration of the pesticidal compound.
The term xe2x80x9cpesticidalxe2x80x9d is not intended to refer only to the ability to kill pests, but also includes the ability to interfere with a pest""s life cycle in any way that results in an overall reduction in the pest population. For example, the term xe2x80x9cpesticidalxe2x80x9d includes inhibition of a pest from progressing from one form to a more mature form, e.g., transition between various larval instars or transition from larva to pupa or pupa to adult. Further, the term xe2x80x9cpesticidalxe2x80x9d is intended to encompass anti-pest activity during all phases of a pest""s life cycle; thus, for example, the term includes larvacidal, ovicidal , and adulticidal activity.
The word xe2x80x9ctransformxe2x80x9d is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including for example, direct transmission of a polynucleotide sequence from a cell or virus particle, transmission by infective virus particles and transmission by any other nucleotide-bearing construct) resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell.
The terms xe2x80x9cpolypeptide,xe2x80x9dxe2x80x9cpeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d as used herein are intended to refer to amino acid sequences of any length.
SEQ ID NO. 1 is the amino acid sequence for TMOF.
SEQ ID NOS. 2-42 are TMOF peptide analogs according to the subject invention.
SEQ ID NOS. 43-44 are TMOF receptors according to the subject invention.
The subject invention concerns novel pest control compounds and methods for using such compounds. Specifically exemplified are novel pesticidal polypeptides, compositions comprising said pesticidal polypeptides and the use of such pesticidal polypeptides and compositions in controlling pests, such as mosquitoes.
In one aspect, the biological control agents comprise novel polypeptides, comprising an amino acid sequence of the formula:
A1A2A3A4A5Fxe2x80x83xe2x80x83(Formula I)
wherein:
A1 is selected from the group consisting of Y, A, D, F, G, M, P, S, and Y;
A2 is selected from the group consisting of A, D, E, F, G, N, P, S, and Y;
A3 is optionally present and is selected from the group consisting of A, D, F, G, L, P, S, and Y;
A4 is optionally present when A3 is present and is selected from the group consisting of A, F, G, L, and Y;
A5 is optionally present when A4 is present and is selected from the group consisting of A, F, L, and P;
F is a flanking region which is optionally present and is selected from the group consisting of P, PP, PPP, PPPP, and PPPPP.
The peptide preferably does not consist of YDPAP6, DYPAP6, PAP6, YDPAP, YDPAP2, YDPAP3, YDPAP4, NPTNLH, or DF-OMe.
In a narrower aspect the polypeptide comprises an amino acid sequence which consists essentially of the amino acid sequence of Formula I. In a preferred aspect, where the amino acid sequence is a TMOF fragment, the peptide or protein lacks TMOF amino acids adjacent to the TMOF fragment. In still another aspect, the pesticidal polypeptide consists of the amino acid sequence of Formula I.
In another aspect, only A1, A2, A3, A4, and F of Formula I are present and F is attached to A4. In yet another aspect, only A1, A2, A3, and A4 of Formula I are present. In an additional aspect, only A1, A2, A3 and F of Formula I are present and F is attached to A3. In another aspect, only A1, A2, and A3 of Formula I are present. In another aspect, only A1, A2, and F of Formula I are present,. and F is attached to A2. In a further aspect, only A1 and A2 of Formula I are present. In a preferred mode, A and D are present in the amino acid sequence and, more preferably, A, D, and Y are present.
One embodiment of the subject invention concerns a peptide having the formula A1A2 (Formula II) wherein A1 is an amino acid selected from the group consisting of A, D, F, M, and Y, and A2 is an amino acid selected from the group consisting of A, D, E, P, and Y. In a preferred embodiment, the subject invention is directed to peptides which comprise the amino acids A, D, and Y.
In a preferred mode, the pesticidal polypeptides of the present invention comprise truncated forms of TMOF having 50% or fewer amino acids than the corresponding TMOF. Preferably the truncated forms have 2-10 amino acid residues. More preferably the truncated forms have 2-8 amino acid residues. Most preferably, the truncated forms have 2-5 amino acid residues. Despite this substantial truncation, the pesticidal polypeptides retain some or all of the biological activity of the full-length peptide. Further, the pesticidal polypeptides of the subject invention are particularly advantageous because of more rapid and efficient penetration into the midgut, and are less expensive to produce by conventional chemical methods.
Preferably, the subject peptides have an LD50 against mosquito larvae of less than 3.0 xcexcmole/ml. More preferably, the peptides have an LD50 of less than 2.0 pmole/ml, and, most preferably, the peptides have an LD50 of less than 1.0 xcexcmole/ml. As used herein, xe2x80x9cLD50xe2x80x9d refers to a lethal dose of a peptide able to cause 50% mortality of larvae maintained on a diet of 1 mg/ml autoclaved yeast supplemented with the pesticidal polypeptide. (Borovsky, Dov, and Farida Mahmood [1995] Regulatory Peptides 57:273-281).
The one-letter symbol for the amino acids used herein is well known in the art. For convenience, the relationship of the three-letter abbreviation and the one-letter symbol for amino acids is as follows:
The pesticidal polypeptides of the present invention may be provided as a fusion polypeptide, the amino acid sequence of which includes one or more pesticidal polypeptides of the present invention, and optionally comprises one ore more heterologous polypeptides. In various specific embodiments, two or more of the pesticidal polypeptides are linked, for example, by peptide bonds between the N-terminus of one portion and the C-terminus of another portion. In other aspects, one or more of the pesticidal polypeptides can be linked to one or more heterologous peptides or proteins to form pesticidal fusion polypeptides. Molecules comprising such portions linked by hydrocarbon linkages are also provided. Derivatives of the foregoing fusion proteins are also provided (e.g., branched, cyclized, or C-terminal chemically modified, etc.). Further, two or more peptides of the general Formulas I and/or II can be linked to each other.
Pesticidal polypeptides comprising the sequences of Formulas I and/or II in which only conservative substitutions have been made are also provided by the present invention. Analogues which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analogue having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally, to provide a sulfhydryl group for disulfide bond formation), are also provided.
Nonclassical amino acids and/or chemical amino acid analogues can be introduced as a substitution for an existing amino acid residue, or as an insertion into the amino acid sequence of the pesticidal polypeptides of the present invention, or as an addition to a terminus of the pesticidal polypeptides. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, xcex1-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, xcex3-Abu, xcex5-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citruline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, xcex2-alanine, fluoro-amino acids, designer amino acids such as xcex2-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). Dextrorotary amino acids are indicated herein by a parenthetical D, i.e., xe2x80x9c(D)xe2x80x9d, immediately preceding the dextrorotary amino acid.
Thus, the pesticidal polypeptide derivatives include peptides containing as a primary amino acid sequence all or part of the peptide sequence of Formulas I and/or II, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence, resulting in a peptide which is functionally active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 1). Such pesticidal polypeptide derivatives can be made either by chemical peptide synthesis or by recombinant production from polynucleotide sequences encoding the pesticidal polypeptide. The subject invention further includes other pesticidal compounds which bind to a TMOF receptor. As used herein, xe2x80x9cTMOF compoundsxe2x80x9d refers to pesticidal polypeptides of Formulas I and II as well as other polypeptides and compounds which bind to a TMOF receptor as described herein.
TMOF receptors and polynucleotides. In one embodiment, the subject invention is directed to the control of pests using a pesticidal compound which binds to or otherwise associates with a TMOF receptor. Specifically exemplified herein is a TMOF receptor comprising the amino acid sequence shown in SEQ ID NO. 44. Preferably, the polypeptide is encoded by a complete polynucleotide nucleotide sequence of a TMOF receptor gene, or a fragment analogue, derivative or other functional equivalent thereof which encodes polypeptides having TMOF receptor activity. In a specific embodiment, the TMOF receptor is encoded by a polynucleotide sequence comprising the coding sequence (nucleotides 1-186) shown in SEQ ID NO. 43 or other polynucleotide sequence with codons encoding the amino acid sequence of SEQ ID NO. 44.
Isolated TMOF receptors can be used to produce antibodies according to known techniques. These antibodies may be monoclonal or polyclonal. These antibodies can be used to screen an expression library to identify other clones expressing polypeptides having TMOF receptor activity. Alternatively, these antibodies may be used to identify TMOF receptors from their natural material such as mosquito gut material.
A specific TMOF receptor sequence is exemplified herein. This sequence is merely exemplary of TMOF receptors. Variant or equivalent receptors (and nucleotide sequences coding for equivalent receptors) having the same or similar TMOF receptor activity can also be utilized. Equivalent receptors will typically have amino acid homology with the exemplified receptor. This amino acid identity will typically be greater than 60%, preferably be greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and can be greater than 95%. These identities are as determined using standard alignment techniques. The amino acid homology will be highest in critical regions of the receptor that account for biological activity or are involved in the determination of three-dimensional configuration, which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions that are not critical to biological activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not completely diminish the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not completely diminish the biological activity of the receptor.
The use of polynucleotide probes is well known to those skilled in the art. In one specific example, a cDNA library for mosquito gut cells can be created by routine means, and DNA of interest can be isolated therefrom. Polynucleotides of the subject invention can be used to hybridize with DNA fragments of the constructed cDNA-library, allowing identification of and selection (or xe2x80x9cprobing outxe2x80x9d) of the genes of interest, i.e., those nucleotide sequences which hybridize with the probes of the subject invention and encode polypeptides having TMOF receptor activity. The isolation of these genes can be performed by a person skilled in the art, having the benefit of the instant disclosure, using techniques which are well-known in the molecular biology art.
Thus, it is possible, without the aid of biological analysis, to identify polynucleotide sequences encoding TMOF receptors. Such a probe analysis provides a rapid method for identifying genes encoding TMOF receptors from a wide variety of hosts. The isolated genes can be inserted into appropriate vehicles which can then be used to transform a suitable host.
Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170.
Examples of various stringency conditions are provided herein. Hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods (Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.). In general, hybridization and subsequent washes can be carried out under moderate to high stringency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25xc2x0 C. below the melting temperature (Tm) of the DNA hybrid in 6xc3x97SSPE, 5xc3x97Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz et al. et al. [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).
Tm=81.5xc2x0 C.+16.6 Log[Na+]+0.41(%G+C)xe2x88x920.61(%formamide)xe2x88x92600/length of duplex in base pairs.
Washes are typically carried out as follows:
(1) twice at room temperature for 15 minutes in 1xc3x97SSPE, 0.1% SDS (low stringency wash);
(2) once at Tm-20xc2x0 C. for 15 minutes in 0.2xc3x97SSPE, 0.1% SDS (moderate stringency wash).
For oligonucleotide probes, hybridization can be carried out overnight at 10-20xc2x0 C. below the melting temperature (Tm) of the hybrid in 6xc3x97SSPE, 5xc3x97Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes can be determined by the following formula:
Tm (xc2x0 C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs et al. [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683-693).
Washes can be carried out as follows:
(1) twice at room temperature for 15 minutes 1xc3x97SSPE, 0.1% SDS (low stringency wash;
(2) once at the hybridization temperature for 15 minutes in 1xc3x97SSPE, 0.1% SDS (moderate stringency wash).
In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment  greater than 70 or so bases in length, the following conditions can be used:
Low: 1 or 2xc3x97SSPE, room temperature
Low: 1 or 2xc3x97SSPE, 42xc2x0 C.
Moderate: 0.2xc3x97 or 1xc3x97SSPE, 65xc2x0 C.
High: 0.1xc3x97SSPE, 65xc2x0 C.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
Identification of pest control compounds. The TMOF receptors can advantageously be used to identify pesticidal compounds and/or confirm or characterize the activity of the pesticidal polypeptides of the subject invention. The pesticidal compounds of the subject invention are preferably those which bind to, or otherwise associate with, the TMOF receptor in a way which activates the TMOF receptor, thereby inhibiting biosynthesis of TTLE, resulting in control of the pest population. A person skilled in the art, having the benefit of the instant disclosure, can utilize the TMOF receptors described herein to identify and characterize pesticidal compounds of the subject invention. In one embodiment, the TMOF receptor can be purified from its natural sources using, for example, antibodies to the TMOF receptor to obtain the purified protein. This purified protein can then be used to identify compounds which bind to the receptor. Compounds thus identified can then be further evaluated using, for example, appropriate bioassays to confirm and/or characterize the pest control activity of the compound.
As an alternative to purifying TMOF receptors from their natural material, recombinant TMOF receptor protein can be expressed in an appropriate recombinant host that has been transformed with a polynucleotide sequence encoding the TMOF receptor. The polynucleotide sequence used to transform the appropriate host may comprise, for example, the polynucleotide coding sequence disclosed in SEQ ID NO. 43. The host may be transformed so as to express the TMOF receptor at the cell surface or, alternatively, the TMOF receptor may be retained intracellularly or secreted into the surrounding media. In any case, the expressed TMOF receptor may be isolated from the recombinant host using techniques known to those skilled in the art. The recombinant purified protein can then be used as described above to identify compounds which bind to the receptor. As an alternative embodiment, the receptor expressed at the surface of the recombinant cell can be used in conjunction with the whole cell to identify compounds which bind to the receptor.
In another embodiment, TMOF receptors of the subject invention can be applied to a chip or other suitable substrate to facilitate high throughput screening of potential pest control compounds.
Once compounds are identified which bind to the TMOF receptor, their pesticidal activity can be confirmed and/or characterized using bioassays known to those skilled in the art. The pesticide compounds of the subject invention can have activity against a variety of pests. These pests include agricultural pests which attack plants as well as pests of animals which attack humans, agricultural animals, domestic animals, and wild animals.
Preparation of novel pest control compounds. The novel pesticidal compounds of the invention can be prepared by well-known synthetic procedures. For example, the pesticidal polypeptides of the present invention can be prepared by the well-known Merrifield solid support method. See Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure, using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing peptide chain anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrenedivinylbenzene copolymer. This method conveniently simplifies the number of procedural manipulations, since removal of the excess reagents at each step is effected simply by washing the polymer.
Alternatively, these peptides can be prepared by use of well-known molecular biology procedures. Polynucleotide sequences encoding the pesticidal polypeptides of the invention can be readily synthesized. These polynucleotide sequences are a further aspect of the subject invention. These polynucleotides can be used to transform prokaryotic and eukaryotic cells, such as bacteria, insect, plant, or fungi cells for synthesis of the peptides of the invention. These polynucleotides may also be employed to modify viruses such as bacteriophages for use as cloning and/or expression vectors. One example of a cell line useful in accordance with the present invention includes, the insect cell line Sf9 (Spodoptera rugiperda), deposit number ATCC CRL 1711, which is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA. An example of a useful virus includes Baculovirus Autographa Californica Nuclear Polyhedrosis Virus AcNPV) which is available from Texas AandM University, Texas Agricultural Experiment Station, College Station, Tex. 77843, and has been described in Smith, G., and M. D. Summers 1978) Virology 89:517-527; and (1979) J. Virology 30:828-838. Other nuclear polyhedrosis viruses (See World Health Organization Technical Report No. 531) such as Spodoptera frugiperda (Sf MNPV), Choristoneura fumiferana (Cf MNPV) (Smith, G., and M. D. Summers [1981] J. Virol. 39:125-137), or Spodoptera littoralis (S1 NPV) (Harrap et al. [1977] Virology 79:14-31) can be used instead of Autographa californica NPV. Other insect cell lines can also be substituted for Spodoptera frugiperda (Sf9), for example, Trichoplusia ni (Volkman, L. E., and M. D. Summers [1975] J. Virol. 16:1630-1637), Spodoptera exigua, Choristoneura fumiferana (Smith, G., and M. D. Summers [1981] J. Virol. 39:125-137) and Spodoptera littoralis (Harrap, K. A. et al. [1977] Virology 79:14-31).
In yet another embodiment, the subject invention is directed to polynucleotides which encode the subject pest control peptides. Polynucleotides can be produced by routine methods known in the art. See S. L. Beaucage and M. H. Caruthers (1981), Tetrahedran Lett. 22:1859. The polynucleotides may usefully be presented as expression vectors or as expression cassettes for insertion into expression vectors.
If desired, the polynucleotides of the present invention can be amplified using PCR. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. et al. (1985) xe2x80x9cEnzymatic Amplification of xcex2-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,xe2x80x9d Science 230:1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3xe2x80x2 ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase results in the amplification of the segment defined by the 5xe2x80x2 ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
PCR primers can be designed from the DNA sequences of the subject invention. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions and insertions (especially additions of nucleotides to the 5xe2x80x2 end) of the exemplified sequences fall within the scope of the subject invention. These PCR primers can be used to amplify genes of interest from a sample. Thus, this is another method by which polynucleotide sequences encoding the subject peptides can be identified and characterized.
The various methods employed in the preparation of plasmids comprising the pesticidal polypeptide encoding polynucleotides of the present invention, and transformation of host organisms are well known in the art and are described, for example, in U.S. Pat. Nos. 5,011,909 and 5,130,253. These patents are incorporated herein by reference. These procedures are also described in Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restrictions enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, e.g., E. coli or plant cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.
Production of Recombinant Hosts. In another embodiment, the subject invention is directed to a prokaryotic or eukaryotic cell transformed with a polynucleotide encoding a polypeptide of Formula I or II. Hosts which may be employed according to techniques well known in the art for the production of the polypeptides of the present invention include unicellular microorganisms, such as prokaryotes, i.e., bacteria; and eukaryotes, such as fungi, including yeasts, algae, protozoa, molds, and the like, as well as plant cells, both in culture or in planta, and animal cells.
Furthermore, virsus may also be modified to comprise a polynucleotide encoding a pesticidal polypeptide according to the present invention. Specific bacteria which are susceptible to transformation include members of the Enterobacteriaceae, such as strains of Escherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis; Pseudomonas; Pneumococcus; Streptococcus; Haemophilus influenzae, and yeasts such as Saccharomyces, among others. In one embodiment of the present invention, the transformed host is Bacillus sphaericus, which is known to be highly specific for control of mosquito larvae. In a preferred embodiment, the host is Bacillus sphaericus, serotype H5a5b, available from Abbott Laboratories as VectoLex CG Biological Larvicide (EPA Reg. No. 275-77).
The polynucleotide sequences of the subject invention can be used to transform a host cell; for example, the polynucleotide sequences can be introduced directly into the genome of the transformable host cell or can first be incorporated into a vector which is then introduced into the host. Exemplary methods of incorporation include transduction by recombinant phage or cosmids, transfection where specially treated host bacterial cells can be caused to take up naked phage chromosomes, and transformation by calcium precipitation. These methods are well known in the art. Exemplary vectors include plasmids, cosmids, and phages.
It is well known in the art that when synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. For purposes of the subject invention, xe2x80x9cfrequency of preferred codon usagexe2x80x9d refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable to limit this analysis to genes that are highly expressed by the host cell.
Thus, in one embodiment of the subject invention, prokaryotic or eukaryotic cells such as bacteria, algae, fungi, plant, or other cells can be genetically engineered, i.e., transformed with polynucleotides encoding the subject peptides to attain desired expression levels of the subject peptides. To provide genes having enhanced expression, the DNA sequence of the gene can be modified to comprise codons preferred by highly expressed genes to attain an A+T content in nucleotide base composition which is substantially that found in the transformed host cell. It is also preferable to form an initiation sequence optimal for the host cell, and to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed genes in the host cell to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression.
Assembly of the polynucleotide sequences of this invention can be performed using standard technology known in the art. For example, a structural gene designed for enhanced expression in a host cell can be assembled within a DNA vector from chemically synthesized oligonucleotide duplex segments. Preferably the DNA vector or construct has a suitable origin of replication; a selectable marker, such as antibiotic resistance or fluorescence; appropriate termination sequences; and an operable promoter.
Furthermore, chimeric toxins may be used according to the subject invention. Methods have been developed for making useful chimeric toxins by combining portions of proteins. The individual polypeptides which are combined to form the pesticidal chimeras need not be pesticidal, so long as the combination of portions creates a chimeric protein which is pesticidal. The chimeric toxins may include portions from toxins which do not necessarily act upon the TMOF receptor, for example, toxins from Bacillus thuringiensis (B.t.). e.g., B.t. israelensis, B.t. tenebrionis, B.t. san diego, B.t. aizawa, BHt. subtoxicus, B.t. alesti, B.t. gallaeriae, B.t. sotto, B.t. kurstaki, B.t. berliner, B.t. tolworthi, B.t. dendrolimus, and B.t. thuringiensis, and delta endotoxins as described in U.S. Pat. No. 5,686,069.
With the teachings provided herein, one skilled in the art can readily produce and use the various toxins and polynucleotide sequences described herein.
The polynucleotide sequences and polypeptides useful according to the subject invention include not only the exemplified sequences but also fragments analogues, derivatives and variants thereof which retain some or all of the characteristic pesticidal activity of the TMOF peptides, or which exhibit improved activity as compared to the activity of the TMOF peptides. As used herein, the terms xe2x80x9cvariantsxe2x80x9d or xe2x80x9cvariationsxe2x80x9d of genes in reference to nucleotide sequences means nucleotide sequences encoding the same peptides or encoding equivalent peptides having pesticidal activity. As used herein, the term xe2x80x9cequivalent peptidesxe2x80x9d refers to peptides having some or all of the same biological activity of the pesticidal peptides.
Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as BAL31 or site-directed mutagenesis can be used to systematically excise nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these peptides.
Polynucleotide sequences encoding the pesticidal polypeptides of the present invention can be introduced into a wide variety of microbial or plant hosts. In the case of toxins, expression of the gene results, directly or indirectly, in the production and maintenance of the pesticide. With suitable microbial hosts, e.g., yeast or Chlorella, the microbes can be applied to the situs of the pest where they will proliferate and be ingested resulting in control of the pest. Alternatively, the microbe hosting the gene can be killed and optionally treated under conditions that retain and/or prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest. In one embodiment, the host is transformed such that the gene encoding the pesticidal polypeptide is only expressed or maintained for a relatively short period of time, such as days or months, so that the material does not persist in the environment.
A wide variety of means are available for introducing a polynucleotide sequence encoding a pesticidal polypeptide into a host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.
Synthetic genes that encode peptides that are functionally equivalent to the toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
Recombinant cells expressing a pest control compound can be treated to prolong the toxin activity and stabilize the cell. For example, the pesticidal polypeptides can be formulated as pesticide microcapsules comprising the pesticidal polypeptide within a cellular structure that has been stabilized and protects the pesticidal polypeptide when the microcapsule is applied to the environment of the target pest. Suitable host cells include either prokaryotes or eukaryotes. As hosts, of particular interest are the prokaryotes and the lower eukaryotes, such as algae and fungi. The cell is preferably intact and substantially in the proliferative form when treated, rather than in a spore form.
Treatment of the microbial cell, e.g., a microbe containing the polynucleotide sequence encoding the pesticidal polypeptide, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not eliminate the pesticidal properties of the pesticidal polypeptide, nor eliminate the cellular capability of protecting the toxin. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462.
Methods and Formulations for Control of Pests. Control of pests using the pest control compounds of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of recombinant microbes to the pests (or their habitats, food sources, etc.), and the transformation of plants with genes (polynucleotide sequences) encoding the pesticidal polypeptides of the subject invention. Transformations can be accomplished by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.
The plant pests that can be controlled by the compounds of the subject invention include pests belonging to the orders Coleoptera, Lepidopterans, Hemiptera and Thysanoptera. These pests all belong to the phylum Arthropod. Other pests that can be controlled according to the subject invention include members of the orders Diptera, Siphonaptera, Hymenoptera and Phthiraptera. Other pests that can be controlled by the compounds of the subject invention include those in the family Arachnida, such as ticks, mites and spiders.
The use of the compounds of the subject invention to control pests can be accomplished readily by those skilled in the art having the benefit of the instant disclosure. For example, the compounds may be encapsulated, incorporated in a granular form, solubilized in water or other appropriate solvent, powdered, and included into any appropriate formulation for direct application to the pest or to a pest-inhabited locus. In a preferred embodiment for the control of plant pests, plants may be genetically transformed to express a pesticidal polypeptide, such that a pest feeding upon the plant will ingest pesticidal polypeptide and thereby be controlled.
Where the polynucleotide sequence is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is preferred that certain host microbes be used. In one aspect, preferred microbes are known to occupy the xe2x80x9cphytospherexe2x80x9d (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest or the situs where the pest proliferates. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type organisms, provide for stable maintenance and expression of the gene expressing the pesticidal polypeptide and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium, and algae, e.g., Chlorella. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. The pigmented microorganisms are preferred.
Formulated bait granules containing an attractant and the pesticidal polypeptides of the present invention, or recombinant microbes comprising toxin-encoding polynucleotide sequences, can be applied to a pest-inhabited locus, such as to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants or polymers.
As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least about 0.0001% by weight and may be 100% by weight. The dry formulations will have from about 0.0001-95% by weight of the pesticide while the liquid formulations will generally be from about 0.0001-60% by weight of the solids in the liquid phase. The formulations that contain cells will generally have from about 1 to about 1010 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling or the like.
In applications to the environment of the target pest, the transformant strain can be applied to the natural habitat of the pest. In some cases,the transformant strain will grow in the pest upon ingestion, while continuing to produce the pesticidal polypiptide(s) The organism may be applied by a wide variety of methods known in the art, including pouring, spraying, soaking, injection into the soil, seed coating, seedling coating or spraying or the like.
In aquatic environments, pest control may be attained at or below the surface by adjusting the specific gravity of the microbe. This can be accomplished by, for example, varying the lipid content of the transformant microorganism strain. It is known that many indigenous aquatic algae float due to their lipid content. A variation in lipid content will allow the transformant strain to be distributed at desired depths below the water surface.
The pest control compounds may also be provided in tablets, pellets, briquettes, bricks, blocks and the like which are formulated to float, maintain a specified depth or sink as desired. In one embodiment the formulations, according to the present invention, are formulated to float on the surface of an aqueous medium; in another embodiment they are formulated to maintain a depth of 0 to 2 feet in an aqueous medium; in yet another embodiment the formulations are formulated to sink in an aqueous environment.
For commercial formulations, the organisms may be maintained in a nutrient medium which maintains selectivity and results in a low rate of proliferation. Various media may be used, such as yeast extract or L-broth. Once the organism is to be used in the field, the non-proliferating concentrate may be introduced into an appropriate selective nutrient medium, grown to high concentration, generally from about 105 to 109 cells/ml and may then be employed for introduction into the environment of the pest.
All of the U.S. patents and other references cited herein are hereby incorporated by reference, as are co-filed U.S. patent application Ser. No. 09/295,846, (UF-223) Transformed Cells Useful for the Control of Pests; U.S. patent application Ser. No. 09/295,849, (UF-216) Neuropeptides and their use for Pest Control; U.S. patent application Ser. No. 09/296,113, (UF-224) Materials and Methods Useful for the Control of Insect Larvae; and U.S. patent application Ser. No. 09/295,924, Compositions and Methods for Controlling Pests.